The public health problems posed by tuberculosis have grown more serious as a consequence of the global AIDS epidemic and the emergence of multidrug resistant strains of M. tuberculosis. To combat this worldwide scourge, vaccine development for tuberculosis is an international priority. Cellular immunity to M. tuberculosis is critical in controlling the infection and both CD4+ and CD8+ T cells are important. Because CD8+ T cells are required for immunity to tuberculosis, there is great interest in vaccine strategies that elicit M. tuberculosis-specific CD8+ T cells. However, evaluating the role of CD8+ T cells in host resistance and vaccine-induced immunity has been hampered because few class I MHC-presented mycobacterial antigens have been identified. Thus, even basic questions concerning the function of CD8+ T cells during M. tuberculosis infection remain unanswered. We have identified an epitope of the CFP10 protein that is recognized by up to 30% of the CD8+ T cells in the lungs of mice following respiratory M. tuberculosis infection. The CFP10 gene is located within the RD1 region of the mycobacterial genome, which is required for the virulence of M. tuberculosis, and it is likely that CFP10 is mycobacterial virulence factor. Furthermore, it is a major target of the immune response and most people infected with M. tuberculosis have evidence of B and T cell immunity to the CFP10 antigen. We propose to investigate the role of CFP10specific CD8+ T cells in immunity to M. tuberculosis. In Aim 1, the molecular basis for in vivo CD8+ T cell mediated cytotoxicity will be established. These results will determine whether CTL primed during M. tuberculosis infection kill target cells by cytotoxic granule exocytosis, the CD95/95L pathway, or by another mechanism. We will also address whether infected macrophages are killed in vivo. Aim 2 addresses whether CFP10-specific CD8+ T cells elicited by vaccination can protect mice against respiratory challenge with M. tuberculosis. CFP10-vaccinated mice will be challenged with M. tuberculosis and how CFP10-specific CD8+ T cells respond following challenge and whether these T cells contribute to host resistance will be determined. Aim 3 will specifically determine whether cytotoxic activity or other IFNg-independent functions are required for the protection mediated by CD8+ T cells against M. tuberculosis. Finally, Aim 4 will address whether CD8+ memory T cells are generated during chronic tuberculosis infection. The related questions of how bacterial persistence affects T cells memory and how pre-existing mycobacterial immunity alters the M. tuberculosis-specific immune response will also be determined. This proposal will directly assess how CFP10-specific CD8+ T cells contribute to host defense and how their function is modulated by immunity and disease. [unreadable] [unreadable]